Water Interface: Studies on

The two synthetic sterol–phospholipid hybrids DCholPC and PCholPC were investigated in monolayers at the air/water interface. Study was based on πâ...
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Sterol−Phospholipid Hybrids at the Air/Water Interface: Studies on Properties and Interactions with Parent Lipid Molecules Michał Flasiński,*,† Beata Konderla,† Marcin Broniatowski,† and Paweł Wydro‡ †

Department of Environmental Chemistry, Faculty of Chemistry, Jagiellonian University, Gronostajowa 3, 30-387 Kraków, Poland Department of Physical Chemistry and Electrochemistry, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland

‡

S Supporting Information *

ABSTRACT: The two synthetic sterol−phospholipid hybrids DCholPC and PCholPC were investigated in monolayers at the air/water interface. Study was based on π−A isotherm analysis complemented with application of grazing incidence X-ray diffraction. It was found that both compounds are capable of forming stable, highly condensed monolayers of a surface characteristics typical for sterols. GIXD studies show that the crystallographic area for DCholPC monolayer is very similar to that found for cholesterol (37.1 vs 38.0 Å2), while for PCholPC (28.8 Å2) it is significantly smaller as compared to area for the mixed Chol/DPPC 2/1 monolayer (33.4 Å2). In our study the problem of interactions between investigated sterol−phospholipid hybrids and natural membrane lipid components was for the first time analyzed in planar lipid systems. Studies on mixed monolayers showed that both hybrids, similarly to cholesterol, reveal a condensing effect toward DPPC acyl chains; however, DCholPC having two steroid moieties in the molecule was found to be more efficient. On the other hand, the sterol moiety and the hydrocarbon chain of PCholPC molecule are packed in the 2D crystalline phase extremely tight. Our studies showed that the investigated compounds can be applied as biocompatible components of stable liposomes.

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INTRODUCTION Properties of Sterol−Phospholipid Hybrids. Modification of the lipid molecules chemical structure is, besides manipulation of the system composition, one of the ways to obtain desirable properties of the artificial membranes applied in scientific investigations as well as in preparation of lipid carriers such as liposomes. It is well-known that vesicles prepared solely from phospholipids may not possess desirable properties, like e.g. enough solubility or half-life, and in particular are not satisfactorily stable, which may lead to too fast or uncontrolled release of the liposome content before reaching the target.1 One way to deal with this problem is to incorporate additional amphipathic, preferentially highly biocompatible molecules into the lipid mixture of the vesicle components.2 Cholesterol or plant sterol molecules fulfill this criterion, since addition of these natural compounds causes reduction of the bilayer permeability, modulates phospholipid chains’ organization, and eliminates phospholipid phase transition, which in turns leads to increase of the vesicle’s stability.3,4 Unfortunately, © XXXX American Chemical Society

incorporation of the sterol into the biomembranes is not deprived of inconvenience, as cholesterol can rapidly transfer between the bilayers, whereas above 50 mol % of its content, it tends to phase separate from the mixed system.5 Another, more sophisticated approach to obtain expected properties of the lipid systems is to perform chemical modification of the molecular structures based on common structural motifs found in naturally occurring molecules.6,7 Among such compounds known from the literature, especially those built on the phospho-8 or sphingolipid9 molecular constructs seem to be the most promising. In these compounds, so-called chimeras (hybrids) or sterol-modified phospholipids, the main structural modifications concern incorporation of the steroid rings system into the sn-1 and/or sn-2 positions. This is frequently realized with application of molecular spacers and linkages possessing Received: November 28, 2015 Revised: March 24, 2016

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DOI: 10.1021/acs.langmuir.5b04311 Langmuir XXXX, XXX, XXX−XXX

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Langmuir ester, ether, or carbamate groups.10 This idea opens a wide perspective of synthesis directed toward receiving products of the most suitable properties. The preliminary experiments revealed that application of sterol-modified phospholipids in production of liposomes improve their properties. For example, Huang and Szoka found that liposomes formed by sterolmodified glycerophospholipids are at physiological temperature exceptionally stable in the presence of serum.10,11 Another study showed that vesicles containing sterol−phospholipid hybrids demonstrate comparable pharmacokinetic and biodistribution patterns as compared to cholesterol/phospholipid liposomes, but in contrast, they reveal a slower rate of encapsulated substance delivery into the target place.8 Moreover, having cholesterol covalently linked to the phospholipid backbone, it is possible to prevent sterol flip flop or its transferring into the other lipid compartments.12−14 In the case of sterol−phospholipid hybrids, the exchange ratio between bilayers was found to be more than 100-fold less than for free cholesterol.10 For our studies two representatives of sterol− phospholipid hybrids were selected. As can be seen in Scheme 1, the investigated compounds possess structural motives characteristic for both sterol and

have not been discussed comprehensively; thus, still a lot of questions remain without answers. There is lack of information about the state and condensation of the hybrid monolayer, and the problem regarding a phase transition has not been analyzed. Also, it is not known how molecules in hybrid monolayer are organized at the air/water interface. Finally, our purpose was to analyze the interactions of sterol−phospholipid hybrids with the main components of lipid membranes, which is of great importance since such synthetic compounds may possess undesirable membrane activity and reveal toxicity toward cells. Such an assumption should be taken into account, since in living organisms cholesterol occurs as a free molecule, cholesterol sulfate, or in the esterified form but never as the sterol-modified molecules of phospho- or sphingolipids.16−18 The main purpose of the undertaken studies was to perform the characteristics of the behavior of sterol−phospholipid hybrids in monolayers at the air/water interface. The key factors vital from the viewpoint of their application as the biologically active substance carriers concern, e.g., the physical state, condensation, stability, and molecular order of their monolayers as well as the influence of temperature onto these properties. Furthermore, as the compounds of potential application in living organisms, the problem of hybrid−membrane lipid interactions is of utmost importance. To shed new light on this fact, we decided to check the influence of the studied synthetic compounds on the monolayers formed by cholesterol and DPPC. Additionally, these membrane lipids were chosen since thanks to the structural similarities, they can be treated as the parent molecules for the investigated sterol−phospholipid hybrids. We believe that our findings will be helpful in the perspective of hybrids application in formulation of stable liposomes or planar lipid bilayer with reduced mobility of sterol components.

Scheme 1. Molecular Structures of the Investigated Sterol− Phospholipid Hybrids: 1,2-Dicholesterylhemisuccinoyl-snglycero-3-phosphocholine, DCholPC (Top) and 1Palmitoyl-2-cholesterylhemisuccinoyl-sn-glycero-3phosphocholine, PCholPC (Bottom)

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EXPERIMENTAL SECTION

Materials. The investigated sterol−phospholipid hybrids DCholPC and PCholPC of high purity (>99%) were purchased from Avanti Polar Lipids and used without further purification. The membrane lipids, i.e., cholesterol and DPPC (1,2-dipalmitoyl-sn-glycero-3phosphocholine), of the highest purity available in stock (>99%) were purchased from Sigma-Aldrich and Avanti Polar Lipids, respectively. Spreading solutions of all lipids applied in the study of the concentration close to 0.20 mg/mL were prepared in chloroform/ methanol 9/1 (v/v) mixture. Chloroform of spectroscopic purity (99.9% stabilized by ethanol) and methanol (99,9%) were provided by Sigma-Aldrich. In all experiments on Langmuir trough ultrapure water of resistivity ≥18.2 MΩ·cm from Milli-Q system was applied as a subphase. Mixed solutions of the declared compositions were prepared from the respective stock solutions of pure lipids. We investigated the following surface proportions of component 1 (hybrid)/component 2 (membrane lipid): 1/4, 1/2, 1/1, 2/1, and 4/1, which led to the following mole ratios of the component 1: X1 = 0.20, 0.33, 0.50, 0.67, and 0.80, respectively. Methods. Langmuir Experiments. In routine experiments, π−A isotherms were recorded with the NIMA (Coventry, U.K.) Langmuir trough of total area of 300 cm2 equipped in single movable barrier placed on antivibration table. The surface pressure was measured with the accuracy of 0.1 mN/m using a Wilhelmy balance equipped with a surface pressure sensor made of filter paper (ashless Whatman). The required amounts of lipid solutions were spread on pure water surface with Hamilton microsyringe precise to 2 μL. In each experiment, the monolayer was left to equilibrate for at least 5 min before the monolayer compression was initiated with the barrier speed of 20 cm2/ min (∼12 Å2/(molecule min)). The circulating water system was used to control subphase temperature. The condensation of the studied surface films were characterized based on the compression modulus,

phospholipid molecules. Namely, the polar part contains the headgroup of phosphatidylcholine (PC) linked to the glycerol backbone. As far as the nonpolar part of the molecule is concerned, in the case of DCholPC two cholesterol substituents are connected with the molecular core via the hemisuccinyl linkage, whereas in PCholPC molecule, in sn-1 position the palmitoyl hydrocarbon chain is attached. Aim of the Study. In the present study, selected hybrids were characterized in monolayers at the air/water interface. The studies on the behavior of sterol−phospholipid hybrids in model lipid systems are crucial from at least two points of view: First of all, it is important to recognize the main properties of these compounds in one component monolayers. It concerns the stability, state, and condensation of their monolayers formed at the air/water interface, the influence of temperature, and other experimental conditions as well as the composition of the aqueous subphase. Such characteristics have never been performed in a comprehensive manner, even though some attempts have been made in the paper by Foglia et al.15 Unfortunately, some of the results concerning monolayer study B

DOI: 10.1021/acs.langmuir.5b04311 Langmuir XXXX, XXX, XXX−XXX

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Figure 1. Bragg peaks (diffracted intensity vs the horizontal scattering vector Qxy) for the monolayers of sterol−phospholipid hybrids: DCholPC (35 mN/m) and PCholPC (30 mN/m). defined as CS−1 = −A(dπ/dA).19 The miscibility of the investigated lipids in mixed Langmuir monolayers and the interactions between components were analyzed quantitatively on the basis of the calculated values of the excess free energy of mixing (ΔGExc), defined as ΔGExc = NA∫ π0A12 − (A1X1 + A2X2) dπ, where A12 is the mean molecular area for a particular composition of a binary film at a given surface pressure, A1 and A2 are the mean molecular areas for pure monolayers of components 1 and 2, respectively, taken at the same surface pressure, X1 and X2 indicate molar fractions of the components in the mixture, and NA is Avogadro’s number.20 Grazing Incidence X-ray Diffraction. GIXD experiments were carried out in SOLEIL synchrotron center (Paris, France) on the liquid surface diffractometer at the SIRIUS beamline. The dedicated Langmuir trough placed in a gastight canister is mounted on the goniometer of the diffractometer. Before each experiment, the canister was sealed and flushed with helium to reduce oxygen level in order to reduce the scattering background and to minimize the beam damage during the experiment. After at least 30 min, a monolayer was compressed to the target surface pressure, which was held constant during the entire experiment. The surface pressure was measured with a Wilhelmy balance (R&K, Germany) equipped with a filter paper stripe as a π sensor. The detailed construction of the diffractometer working at SIRIUS beamline and the parameters of the synchrotron beam applied in the GIXD experiments are described at the SOLEIL Web site (www.synchrotron-soleil.fr) as well as in the previous papers.21,22

Table 1. Structural Parameters Calculated for Investigated Monolayers from GIXD Data composition of monolayer Chol 30 mN/ ma DCholPC 35 mN/m DPPCa

PCholPC 30 mN/m Chol/DPPC 2/1 a

Bragg peak Qxy [Å−1] 1.094 1.108 ⟨−1,1⟩ 1.467 ⟨1,0⟩ 1.393 ⟨0,1⟩ 1.352 1.258 1.168

lattice parameters [Å, Å, deg]

area [Å2]

a = b = 6.628 γ = 120 a = b = 6.548 γ = 120 a = 4.990 b = 5.142 γ = 115.4 a = b = 5.767 γ = 120 a = b = 6.211 γ = 120

38.0

38

37.1

38

23.2

28.8

L⟨−1,1⟩ 271 L⟨1,0⟩ 50 L⟨0,1⟩ 68 27

33.4

34

Lxy [Å]

Data taken from ref 23.

For example, the unit cell area is larger only of 0.9 Å2, which additionally can be connected with small difference of the surface pressures for both monolayers. It is worth mentioning that the width of the peaks recorded for cholesterol and DCholPC monolayers are very similar; therefore, the average diameters of periodically ordered domains are in both surface films the same (the coherence length in the direction of the diffraction vector is equal to 38 Å). In the case of PCholPC surface film, the registered signal is noticeably wider and its maximum is shifted to 1.258 Å−1. This corresponds to the lattice parameter a = 5.767 Å−1 and the unit cell area of 28.8 Å2. Wider Bragg peak indicates that the coherence length in the xy plane is even smaller as compared to the DCholPC and is equal to 27 Å. On the basis of GIXD results, it can be found that the differences in the molecular structures of both investigated sterol−phospholipid hybrids cause significant discrepancies of 2D periodically ordered domains. Since in the case of PCholPC molecule, the mutual proportion of phospholipid hydrocarbon chain to sterol moiety is 1:1, we decided to model this ratio of structural moieties by forming the mixed cholesterol/DPPC. The obtained GIXD results are shown in the Supporting Information, whereas the unit cell parameters are presented in Table 1. In order to compare GIXD results obtained for monolayers of the investigated sterol−phospholipid hybrids with DPPC, being the molecule structurally related to the studied compounds, the structural parameters for the monolayer of

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RESULTS One-Component Langmuir Monolayers. In order to get insight into the molecular organization of sterol−phospholipid hybrids in Langmuir monolayer at the angstrom scale, we decided to apply the grazing incidence X-ray diffraction technique (GIXD). The experiments were carried out for both monolayers at relatively high surface pressure, i.e., 30 and 35 mN/m for PCholPC and DCholPC monolayer, respectively. Such values of the surface pressures were chosen, as in both cases they correspond to the monolayer state of high condensation (solid state). As can be seen in Figure 1, for both monolayers, a single diffraction peak localized in the horizon (i.e., at Qz = 0 Å−1) was measured. In the case of DCholPC, this signal possesses its maximum at 1.108 Å−1, which corresponds to the hexagonal lattice, with the unit cell parameter a = 6.548 Å and the crystallographic area of 37.1 Å2. Interestingly, as can be found in Table 1, these values are very similar to the parameters obtained for the monolayer formed by cholesterol molecules. C

DOI: 10.1021/acs.langmuir.5b04311 Langmuir XXXX, XXX, XXX−XXX

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Figure 2. Surface pressure (π)−mean molecular area (A) isotherms registered for two-component mixed monolayers of the investigated lipid hybrids: DCholPC and PCholPC with cholesterol and DPPC.

Figure 2, the isotherm registered for mixed monolayers containing cholesterol lay between the curves recorded for one-component films. In both systems Chol/DCholPC and Chol/PCholPC, the π−A isotherms for mixed monolayers containing increasing content of the hybrids move toward larger molecular areas as compared to the curve obtained for cholesterol monolayer. Namely, in the case of hybrid molar fraction of 0.2, the mean molecular area in the mixed monolayers is 55 and 49 Å2/molecule for DCholPC and PCholPC, respectively, whereas in the case of molar fraction of 0.8 these values equal 94 and 69 Å2/molecule. In the case of Chol/DCholPC mixed film it can be found that the phase transition observed as an inflection in the course of the isotherm for pure DCholPC film moves from 26 mN/m toward lower surface pressures with the increasing amount of cholesterol in the mixture. On the other hand, the course of the isotherms recorded for Chol/PCholPC monolayers does not change so significantly. For DPPC/DCholPC mixed films the recorded isotherms, with the exception of XDCholPC = 0.8, are at low surface pressure (